(a) Field of the Invention
The invention relates to methods for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production.
(b) Description of Prior Art
For many crops, hybrids, formed by crossing two or more different strains, provide higher yields than do the parental strains themselves. For example, in canola (Brassica napus, campestris), Canada""s most important crop, manually produced hybrids have yields that can be up to 50% greater than those of the parental lines. This phenomenon, termed hybrid vigor, has been most successfully applied in maize (Zea mays). Hybrid maize constitutes approximately 90% of the North American crop.
To produce hybrid seed on a commercial scale, it is necessary to prevent self-pollination of the seed parent of the hybrid cross. This is a relatively simple matter in maize, because the male or pollen-producing organs (the stamens) are located in a different part of the plant than the female, or seed-bearing organs (the carpels), and the stamens, which collectively form a structure called the tassel, can easily be removed manually from the large numbers of plants used in a seed production operation. By contrast, in most crop species, such as canola, the stamens and carpels are located in the same floral structure, and manual emasculation of large numbers of plants is not possible. Hence, seed producers require alternative methods of pollination control for producing hybrids in these crops. A review of the technology developed for this purpose can be found in The PBI Bulletin article of Arnison P. (Arnison P. et al., The PBI Bulletin, January 1997, 1-11).
One alternative method is to use chemicals, called gametocides, that specifically kill pollen. These chemicals are generally expensive and are often only partially effective. For most crops, especially those like canola that flower over an extended period, this type of pollination control is not cost-effective. Nearly all hybrid seed production systems, therefore, rely on genetic control of pollination. Genetic pollination control mechanisms fall into three categories: self-incompatibility, xe2x80x9cmolecular hybridizationxe2x80x9d methods, and cytoplasmic male sterility.
Self incompatibility (SI) results from the capacity of some plants to reject their own pollen. Plants expressing SI can be used as the female line in hybrid production, but, because these plants normally reject their own pollen, it is usually difficult, and cost-ineffective, to propagate or maintain such lines. In addition, self-incompatibility is not found in most plant species. Molecular hybridization methods are the most recently developed. They rely on genetically engineered male sterility, caused by the specific expression of toxic proteins in pollen forming cells. Such introduced toxin genes act as dominant male sterility genes and can be used to generate female lines for hybrid seed production. As with SI, the propagation of such female lines is not straightforward and involves loss of plant material. The usefulness of these methods is still unclear, and at present they are used in the production of only a few hybrid varieties which are not widely grown.
Cytoplasmic male sterility (CMS), is a widespread and classic non-Mendelian trait. CMS plants are incapable of self-pollination and hence when a CMS line is planted alongside a male-fertile line, all the seed that forms on the sterile plants will be a hybrid of the two parents. Unlike most traits, CMS is maternally transmitted, i.e., it is passed on to offspring only through the seed parent. This property results from the fact that the gene or genes that determine CMS are located on mitochondrial DNA (mtDNA). Unlike most genes, which reside in nuclear DNA, genes in mtDNA are transmitted solely through the female in most plant species. As a result of this property of maternal transmission, it is possible to easily propagate female CMS lines, by pollinating with a male fertile xe2x80x9cmaintainerxe2x80x9d line, that is identical to the CMS line in its nuclear genes, but which is male fertile because it lacks the CMS-causing mtDNA. However, again as a result of the maternal transmission of CMS, hybrid plants produced using female CMS lines would also be male sterile because they would carry the male-sterility conferring mtDNA. This is problematic for seed crops such as maize and canola, which require pollen production for the formation of seed, the harvested product. Fortunately, in many crop species, specific dominant nuclear genes termed restorers of fertility (Rf) have been identified that can suppress the male-sterile phenotype and xe2x80x9crestorexe2x80x9d fertility to F1 hybrids. The components of a CMS system, therefore, consist of the CMS line (which contains the male sterile (S) cytoplasm (or mtDNA) and is homozygous for the recessive or maintainer allele of the restorer gene), the maintainer line (which contains a fertile or normal mtDNA (F) but is isogenic with the CMS line at nuclear genetic loci), and the restorer line (which usually contains the male sterile mtDNA but is homozygous for the dominant nuclear Rf gene). The use of these components in a hybrid seed production scheme is illustrated in FIG. 1.
To produce a diverse set of hybrids using CMS , adequate numbers of restorer lines, that contain Rf genes, as well as xe2x80x9cmaintainerxe2x80x9d lines, that are sterilized by the CMS mtDNA, must be available. The development of these lines through conventional genetics is a slow process that minimally requires several years of effort. For example, to develop a new restorer line it is necessary to first cross the recipient line with an existing restorer line to introduce the Rf allele. A series of backcrosses are then required to recover the genotype of the recipient line. Even after many generations of backcrosses some donor DNA linked to the Rf gene will remain, a phenomenon termed linkage drag; this donor DNA may carry deleterious traits and compromise the quality of the recipient strain.
Two CMS systems are proving to have some limited use in hybrid canola seed production. The Polima or pol cytoplasm is capable of conferring male sterility on many canola cultivars, and effective restorer lines, possessing dominant alleles at a single nuclear restorer locus, have been identified. The cost-effectiveness of hybrid production using this system is limited by the fact that pol-induced male sterility tends to be incomplete or xe2x80x9cleakyxe2x80x9d, especially when the female line is exposed to warmer growing conditions. As a result, pol hybrid seed may be contaminated with CMS seeds that result from self-pollination of the female line. As these CMS plants are male sterile and do not spontaneously set seed, their presence can considerably lower the overall yield of the xe2x80x9chybridxe2x80x9d mixture. A second CMS system is based on the use of a hybrid cytoplasm in which the male sterility determinant is derived from a radish cytoplasm termed Ogura or ogu. Male sterility conferred by the ogu cytoplasm is complete. Restorer lines for this system, which were developed by intro-gressing a single radish restorer gene (which we designate here as Rfo) into Brassica napus, have recently become available, but the development of restorer lines is hampered by xe2x80x9clinkage dragxe2x80x9d between the restorer gene and genes that have a negative effect on seed quality.
It would be highly desirable to be provided with methods for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production.
One aim of the present invention is to provide methods for enhancement of naturally occurring cytoplasmic male sterility and for restoration of male fertility and uses thereof in hybrid crop production.
In accordance with the present invention there is provided a method for enhancing naturally occurring cytoplasmic male sterility in plants which comprises the steps of:
a) introducing into the nucleus of a plant cell a gene construct essentially consisting of a sequence encoding a mitochondrial transit peptide fused upstream of and in frame with, at least one of, an unedited form of atp6 gene and an orf224 gene of Brassica napus mitochondria;
b) selecting for plant cells that have acquired the gene construct in step a); and
c) inducing regeneration of selected plant cells to produce a mature plant.
In accordance with the present invention there is also provided a method for restoration of male fertility to cytoplasmic male sterile plants which comprises the steps of:
a) introducing into the nucleus of a plant cell a gene construct essentially consisting of a sequence encoding a mitochondrial transit peptide fused upstream of and in frame with an edited form of a normal mitochondrial gene that is co-transcribed with an unusual CMS-associated mitochondrial gene;
b) selecting for plant cells that have acquired the gene construct in step a); and
c) inducing regeneration of selected plant cells to produce a mature plant.
The preferred plant is Brassica napus. 
Preferably, step b) is effected using a plant transformation vector.
In accordance with the present invention there is also provided a method for restoration of male fertility to polima cytoplasmic male sterile B. napus which comprises the steps of:
a) introducing into the nucleus of a B. napus plant cell a gene construct essentially consisting of a sequence encoding a mitochondrial transit peptide fused upstream of and in frame with an edited form of an atp6 gene of B. napus mitochondria;
b) selecting for plant cells that have acquired the gene construct in step a); and
c) inducing regeneration of selected plant cells to produce a mature plant.
Our results have implications with respect to the practical implementation of pol CMS for the production of hybrid canola and other Brassica crops. The introduction, into pol CMS plants, of genetic constructs that allow the targeting of the product of the edited form of the Brassica atp6 gene to the mitochondria represents a new type of process by which male fertility restored plants can be recovered. This should allow the production of new pol CMS restorer lines in a single step, through plant transformation. This would represent a considerable savings in time and money over the conventional plant breeding methods for generating such lines, which is at present, the only way in which these lines can be generated. In addition, the introduction of constructs that allow the targeting of the products of the unedited form of the atp6 gene or orf224 into the mitochondria to produce plants with enhanced male sterility represents a process through which new pol CMS lines with enhanced maintenance of male sterility could potentially be produced. As mentioned above, the leakiness of pol male sterility is the major impediment to its more widespread use in hybrid canola production and there is no effective means of addressing this. It is also possible that these processes could represent examples of a more general strategy for the production of varieties that enhance the maintenance or that restore CMS in other CMS systems and with other plant species.